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Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils
University of Copenhagen, Denmark.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, no 11443Article in journal (Refereed) Published
Abstract [en]

Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.

Place, publisher, year, edition, pages
Nature Publishing Group: Open Access Journals - Option C / Nature Publishing Group , 2015. Vol. 5, no 11443
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:liu:diva-120227DOI: 10.1038/srep11443ISI: 000356857900001PubMedID: 26108284OAI: oai:DiVA.org:liu-120227DiVA: diva2:842642
Note

Funding Agencies|This work was funded by the Danish Council for Independent Research, Medical Sciences; Danish Council for Independent Research, Medical Sciences, Sapere Aude programme; Drug Research Academy; DANSCATT; Swedish Research Council; Linux cluster at University of Copenhagen by the Carlsberg Research Foundation; FTIR spectrophotometer by the Apotekerfonden

Available from: 2015-07-21 Created: 2015-07-20 Last updated: 2017-12-04
In thesis
1. Studies on molecular aspects of Transthyretin Amyloidosis
Open this publication in new window or tab >>Studies on molecular aspects of Transthyretin Amyloidosis
2015 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are versatile molecules that play a variety of roles in maintaining the human body, e.g. transport of nutrients. Transthyretin (TTR) is a 55 kDa homotetrameric protein found in human plasma and in the cerebrospinal fluid, responsible for the transport of retinol (vitamin A) and T4 (thyroxine). This protein is probably not essential for life, since TTR knockout mice have normal fetal development and lifespan. TTR, like 25 other human proteins, has been associated to the deposition of amyloid aggregates. Previous research has shown that mutations considerably increase the propensity of the protein to form aggregates. However, the wild type protein also exhibits this ability to aggregate, giving rise to the senile systemic amyloidosis disease that affects 20% people over 80 years of age. It is well accepted that self-association of monomeric subunits triggers the disease through tetramer dissociation, since stabilization of the quaternary structure suppresses aggregate formation.

However, a detailed description of the self-assembly mechanism and fibril structure remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state.

In our efforts to study both native and protofibrillar TTR, we realized the need for development of a fluorescent small molecule capable of binding native and protofibrillar TTR, providing distinguishable emission spectra. We used microwave heating for efficient synthesis and fluorescence spectral screening of compounds. We synthesized and tested 22 analogs displaying a variety of functional groups, most of them linked to a stilbene scaffold. We successfully developed two compounds that detect both TTR states at physiological concentrations. The compounds bound with nM-μM affinities and displayed very distinct emission maxima upon binding native or protofibrillar TTR (> 100 nm difference).

We expect these new findings regarding protofibril self-assembly mechanism, together with our novel molecules serve as important tools in future studies of TTR amyloid formation.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 37 p.
Series
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1740
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-122843 (URN)978-91-7685-866-0 (ISBN)
Available from: 2015-11-26 Created: 2015-11-26 Last updated: 2015-11-26Bibliographically approved

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Campos Melo, Raul IvanHammarström, Per

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